Noncovalent dimerization of ubiquitin

Abstract

Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5 mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.

Figure 1

a) A representative region of 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled ubiquitin, collected at concentrations from 0.2 mm to 3.3 mm (rainbow-colored from red to purple, respectively) at 30 °C; b) cartoon and surface representation of ubiquitin. Residues displaying large chemical shift changes (Δω_max≥25 Hz) are colored red. Lys11, Lys48, and Lys63 are shown as ball-and-stick representations; c) changes of chemical shift values over protein concentrations for all perturbed residues can be globally fitted to a monomer–dimer equilibrium with K_D=(4.9±0.3) mm. Relative to the lowest protein concentration (0.2 mm), the chemical shift differences are expressed as (δ_H+δ_N)^1/2, in which δ_H and δ_N are in Hz; d) protein rotational correlation time τ_c can be fitted to the same equilibrium. Error bars represent one standard deviation.

Figure 2

Sedimentation equilibrium analysis of ubiquitin: a) on a 0.8 mm sample with a rotor speed of 20 000 rpm, and b) on a 0.4 mm sample with a rotor speed of 32 000 rpm. The curves can be globally fitted to a monomer–dimer equilibrium model, with K_D=(4.4±1.5) mm. The experimental data are shown as red circles, the fitted curves as black lines. The theoretical sedimentation curves expected for pure ubiquitin monomers are shown as blue lines.

Figure 3

Intermolecular ¹H transverse PRE G2 rates measured on an equimolar mixture (0.5 mm each) of ¹⁵N-labeled wild-type ubiquitin and unlabeled ubiquitin mutant (K11C, K48C, or K63C) conjugated with a maleimide-EDTA-Mn²⁺ probe. Back-calculated PRE rates for residues 1–71 are shown as blue lines. Insets: residues with observed PRE values >20 s⁻¹ are colored purple on protein surface. Error bars represent one standard deviation.

Figure 4

Ensemble structure of the noncovalent ubiquitin dimer; a) the PRE Q factors for all three tagging sites as a function of the number of conformers in the ensemble; b) correlations between observed and calculated PRE values, error bars represent one standard deviation; c) decrease in the relative solvent exposure upon dimerization; residues that are completely buried in the free form (solvent accessible area <10 Å) are denoted with a gray bar; d) reweighed atomic probability map plotted at 20 % threshold for the distribution of the ¹⁵N-labeled ubiquitin (gray meshes) relative to the unlabeled, paramagnetically tagged ubiquitin (purple surface). The two perspectives are related by an 180° rotation. The dimer interface is colored in red, encompassing residues 4–12, 42–51, and 62–71.

Figure 5

Comparison between the crystal structure of Lys48-linked di-ubiquitin and the ensemble structure of ubiquitin noncovalent dimer; a) the proximal unit (purple surface) of the di-ubiquitin crystal structure is superimposed to one subunit in the ensemble structure of the noncovalent dimer; the distal unit is shown as blue cartoon. The noncovalent dimer is represented the same way as in Figure 3; b) colored in orange, a large portion of the noncovalent dimer interface becomes exposed in Lys48-linked di-ubiquitin. The covalent dimer interface is colored in red.

Figure 6

a) Scheme proposed for quaternary dynamics of di- or poly(ubiquitin); a) with an isopeptide linkage, the two adjacent ubiquitin subunits can either adopt open or closed conformations; b) when in a closed state, the two subunits can fluctuate among various relative orientations.